Biogenic Magnetic Nanoparticles From Burkholderia Sp.YN01 Exhibiting Intrinsic Mimic Properties And Their Applications

In recent years,magnetic nanoparticles have attracted considerable attention due to their unique magnetic properties and their enzyme-like catalytic activities.But artificial synthetic magnetic nanoparticles are easy to gather,have poor biocompatibility,high environmental cost in the synthetic process,which limit their development and application.Microbial synthesis method of nanometer particles has got more attention because it can eliminate or reduce the toxicity of raw materials and by-products in the process of' synthesis.Microbial synthesis of magnetic nanoparticles is a kind of-green,environmental friendly new magnetic nanomaterials synthesis strategy,refers to that the magnetic nanoparticles which are called biogenic magnetic nanoparticles(BMNPs)are synthesized through intracellular or extracellular biomineralization using some microorganisms such as bacteria,fungi and actinomycete in the condition of nonnal temperature and pressure.In this thesis,aiming at the shortcomings of the artificial magnetic nanoparticles(AMNPs),we studied microbial synthesis of magnetic nanoparticles,their enzyme properties and application.A new bacteria strain producing magnetic nanoparticles was screened from environmental samples and identified,the effect of growth condition on the strain and its intracellular BMNPs formation was studied,BMNPs were purified from bacteria cells,and were characterized to acquire their composition and magnetic characteristics,their peroxidase-like and catalase-like catalytic properties and mechanisms were investigated,the BMNPs were applied in H2O2,glucose detection and organic pollutants degradation based on their enzyme-like catalytic activities.A novel bacterial strain containing biogenic magnetic nanoparticles(BMNPs)lived in micro aerobic conditions was isolated from the sediments of Songhua River in Harbin,China,and was named as Burkholderia sp.YNO1 through physiological,biochemical and 16S rDNA identification.This is the first report concerning on biogenic Fe3O4 NPs produced in Burkholderia genus.The influences of pH,oxygen concentration,carbon sources,nitrogen sources,iron sources,temperature and culture time on the growth of strain YNO1 and intracellular synthesis of magnetic nanoparticles were studied.The optimal pH was 6.7.When the oxygen concentration was greater than 12%in the cultivation system,the strain YNO1 grew faster,but the yield of magnetic cells was close to 0,the optimal range of oxygen concentration was 0-6%.The optimal carbon source for YNO1 growth and magnetic cells production was the mixture carbon source of succinic acid and tartaric acid with their concention of about 500 mg/L.The optimal nitrogen source was sodium nitrate with the concentration of 150 mg/L.The optimal iron source was quinic acid iron with the concentration between 20 to 25 NM,the optimum temperature was 30?.When the strain YNO1 was cultured in optimal growth medium,the size of the intracellular magnetic nanoparticles,and the length of the bacteria themselves were increasing along with the extension of incubation time,when the incubation time was 2-3 d,the strain growth was in good condition,and the particle size of intracellular magnetic nanoparticles was between 75-150 nm.When incubation time was arrived at 7 d,the bacteria cells went into a state of decay,vision more pieces of bacteria cells,intracellular magnetic nanoparticles size was larger,which could reach 300 nm.The magnetic nanoparticles in bacterial cells were separated and purified following procedures of ultrasonication,ultracentrifugation and magnet adsorption.The BMNPs productivity is about 1.89 mg/Lgrowth medium.Extracted BMNPs from YNO1 were characterized as pure face-centered cubic Fe3O4 with an average size of 80nm through transmission electron microscope(TEM),X-ray diffraction(XRD)and X-ray photoelectron spectroscopy(XPS).The hysteresis parameters of the BMNPs samples such as Coercive force(Be),remanence coercive force(BCr)and the ratio of saturated magnetization(Mrs/Ms)were deduced as 35.6 mT,43.2 mT and 0.47 respectively using physical property measurement system(PPMS),indicating that the BMNPs belong to single magnetic domain crystals and exhibit a ferromagnetic behavior.These results were consistant with the reported magnetic properties of biogenic magnetic nanoparticles in previous studies.Intracellular BMNPs produced from YN01 possessed intrinsic peroxidase-like catalytic activity,its catalytic ability had a linear relation with BMNPs concentration,ane was dependent on pH and temperature,the catalytic ability was stronger under acid condition.BMNPs catalytic kinetics was in accordance with the typical Michaelis Menten equation.the BMNPs have a higher affinity for TMB than that of HRP.The catalytic reaction mechanisms were discussed through electrochemical experiments and electron paramagnetic resonance spectroscopy analysis(ESR),ESR results showed that the BMNPs could catalyze H2O2 to produce hydroxyl radicals,and the origin of peroxidase-like activity is also associated with their ability to transfer electron between electrode and H2O2 according to the electrochemical study.As a novel peroxidase mimetic,the BMNPs were employed to offer a simple,sensitive and selective colorimetric method for H2O2 and glucose determination,The absorbance at 652 nm is proportional to H2O2 concentrations from 0.01 to 8 mM with a detection limit(DL)of 0.005 mM,the linear range for glucose is from 0.01 to 5 mM and the DL is 0.005 mM.The BMNPs could efficiently catalyze the degradation of phenol and congo red dye,when the BMNPs had been reused for seven times,the degradation rate of both phenol and congo red could also maintained at a higher level.The catalytic activity and the application of the BMNPs as catalase mimics were fistly studied.When the pH was above 7,the BMNPs extracted from YN01 were found to possess intrinsic catalase-like activity.The influence factors of catalase-like activity,catalytic kinetics and stability were studied.The results showed that the BMNPs as a catalase mimic were dependent on BMNPs concentration,pH and temperature,the catalytic ability increased gradually with the increase of BMNPs concentration,pH and temperature.Comparing with the natural catalase,BMNPs had high stability under high pH and high temperature,and were not easy to suffer the effect of antibacterial agent sodium azide.The michaelis constant of BMNPs was less than that of natural catalase,indicating that the affinity of BMNPs to the substrate H2O2 is higher.The catalytic constant Kcat and Kcat/Km ratio of BMNPs were closed to that of natural catalase,showing that BMNPs had higher catalase-like activity.Based on their ability of electrochemical reduction of H2O2,electrochemical sensor was prepared,and BMNPs were applied in the electrochemical detection of H2O2.The linear range of detection was 10 ?mol/L-4 mmol/L with a detection limit of 5 ?mol/L.It was found for the first time that Fe3O4 magnetic nanoparticles possess another important antioxidant enzyme SOD activity besides their intrinsic POD and CAT like activities.Though inhibiting the expression of intracellular superoxide dismutase(SOD)of YN01 by adding 2-methoxy estradiol(2-ME),Fe3O4 nanoparticles were found to have the capacity to eliminate the toxic effects of iron and play the inherent SOD like activity to scavenge ROS.This is the first reported concerning on a new physiological functions of bacterial-genic Fe3O4 magnetic nanoparticles.In order to study the physiological functions of CAT-like activity of BMNPs,amino phenyl benzoyl hydrazine(ABAH)was used to inhibit the intracellular POD activity of YNO1.After POD inhibition,H2O2 content increased and accumulated rapidly,but then the accumulation of H2O2 in the cell became gradually less.At the same time,there was the bubble produced in the culture,and BMNPs yield decreased after inhibition of 72 h.It was analyzed that BMNPs played CAT activity to decompose the intracellular accumulation of H2O2 after POD inhibition,then O2 was produced,and the increasing of O2 content in the system resulted in an increase in the low yield of BMNPs.This is also the first report on the physiological functions of eliminating H2O2 by biogenic Fe3O4 magnetic nanoparticles with CAT-like activity in the bacterial cell.